Molecular dynamics simulation analysis of His226 mutation on the dynamics of the ATPase domain of DNaK

Hsp70s are evolutionarily highly conserved ATP-dependent molecular chaperones which are ubiquitously expressed in the cell. They are found in three domains of life and have essential roles in cells, such as aiding proper folding of nascent polypeptides, prevention of polypeptide chains from misfolding and translocation of proteins across membranes. The diverse cellular functions of Hsp70s are based on the recognition of hydrophobic sequences of client protein. Hsp70s corporate with other co-chaperones like nucleotide exchange factors and J-domain proteins. In our study, we used DnaK which is an Escherichia coli homolog of Hsp70. DnaK have an N-terminal nucleotide-binding ATPase domain (NBD) and a C-terminal substrate-binding domain (SBD). These two domains are connected by a highly conserved hydrophobic interdomain linker. There is an allosteric communication between the domains via the hydrophobic linker. Substrate affinity is regulated by ATP binding and hydrolysis, which results in conformational changes in both domains, while ATP hydrolysis is stimulated by substrate binding. In 2007, Swain et al. revealed that the conserved hydrophobic 389VLLL392 sequence of the interdomain linker is responsible for the allosteric communication between NBD and SBD. According to this study, DnaK (1-392) behaves like the substrate-stimulated DnaK that is pH-dependent, and shows higher activity than that of the unstimulated fulllength protein. In contrast, DnaK (1-388) mimics the activity of the substrate-free form of the full-length DnaK. Which amino acids in the catalytic site are responsible in allosteric communication and pH-dependent ATPase activity in the presence of linker are not enlightened so far, however there are several research trying to find out the key residues and reveal the detailed mechanism of DnaK. In this study, the effect of linker 389VLLL392 on the ATP-bound protein conformation and H226A mutation on ATP-bound DnaK's (1-392) construct were investigated by using molecular dynamics simulations. MD simulation trajectories were analyzed by root mean square deviation (RMSD) and, root mean square fluctuation (RMSF) analysis, also by distance measurement in a time-dependent manner and, principle component analysis. From these analysis it was found that distance between the helices which contain His226 and its neighbour helix is closer to each other in DnaK (1-388) constructs. Moreover, this study reveals that His226 contributes the stabilization of residue Thr199 which is suspicious as a phosphate acceptor after the hydrolysis of ATP.