Exploring Protein Interactions Using Orthogonal Space Tempering

The orthogonal space tempering (OST) scheme is a robust and high-order generalized ensemble sampling method that can make sampling in MD simulation much more efficiently and recover the desired thermodynamic properties of interest in complex biophysical protein behaviors. The OST technique provides a powerful tool to study the protein dynamics and mechanistic details of biophysical and biochemical processes that are difficult to be realized by experimental techniques. Here the orthogonal space tempering technique is applied to explore protein dynamics and interactions of actin monomer and TrmD methyltransferase. The first system studied is nucleotide dependent dynamics of actin monomer. Nucleotide-dependent dynamics transition of actin has been studied for a decade. However, those models proposed were based on crystal structures of monomer actin that are typically modified with mutation or complexed with blocking agents to prevent polymerization, and thus subject to alteration. Additionally, typically experimental techniques cannot observe the mechanistic transition in a time-dependent manner. Here dynamics of monomer actin with different states of bound nucleotide—ATP, ADP, and apo form of actin—are investigated. Corresponding conformations and nucleotide binding cleft width-dependent free-energy profiles are calculated via all-atom simulations based on OST scheme. Current results suggest G-actin-ATP prefers flat and closed conformation, while G-actin-ADP and G-actin-APO prefers open conformation. Folding and unfolding motion of D-loop in subdomain 2 is observed in both G-actin with ADP and ATP bound simulations. The dynamic conformations of G-actin-ATP are consistent with previous experimental results. Our simulations will provide insights for the nucleotide-dependent mechanistic transitions and native structure of G-actin with ADP bound after the free energy profiles converge. The second system of interest is the dynamics of TrmD tRNA (m1G37) methyltransferase. m1G37 methylation prevent frameshift errors in translation and is important for bacteria growth. TrmD methyltransferase has been considered as an important drug target. Asymmetric features have been reported in TrmD catalysis and in the TrmD structure with sinefungin and tRNA bound. Yet mechanistic details of TrmD catalysis remains elusive since TrmD with AdoMet and tRNA bound ternary structure has not been resolved. Here free energy calculations based on OST scheme are performed on corresponding conformational changes with perturbation of defined AdoMet dihedral angle on both active and inactive side of TrmD dimer. The conformation of AdoMet ligands and how both sides of TrmD dimer coordinate with each other are investigated. Current results suggest the cooperativity of TrmD dimer is not through AdoMet ligands on both sides but through the positioning of catalytic residues instead. Another Mg[superscript 2+] dependent TrmD methylation simulation perturbing the distance between the target guanosine G37 and Mg[superscript 2+] is performed to study the function of Mg[superscript 2+] in the catalysis. It is found that G37 base moves out the catalytic binding pocket while Mg[superscript 2+] approaches the carbonyl group of G37, which suggests Mg[superscript 2+] is not involved in the transition state during methylation. Further, conserved Mg[superscript 2+] binding motif (residue 162-179) is found in the disorder linker loop. In the simulation, Mg[superscript 2+] binding motif interacts with G37 via Mg[superscript 2+] and water hydrogen bonding network while G37 is moving into the binding site. Those results formulate a new hypothesis that Mg[superscript 2+] and Mg[superscript 2+] binding motif helps with flipping and positioning of G37 into the catalytic binding pocket. After simulations complete, converged free energy profiles will test this new hypothesis and disclose the actual mechanism of TrmD methylation catalysis.