Mg²⁺-Dependent Methyl Transfer by a Knotted Protein: A Molecular Dynamics Simulation and Quantum Mechanics Study

Abstract

Mg 2+ is required for the catalytic activity of TrmD, a bacteria-specific methyltransferase that is made up of a protein topological knot-fold, to synthesize methylated m 1 G37-tRNA to support life. However, neither the location of Mg 2+ in the structure of TrmD nor its role in the catalytic mechanism is known. Using molecular dynamics (MD) simulations, we identify a plausible Mg 2+ binding pocket within the active site of the enzyme, wherein the ion is coordinated by two aspartates and a glutamate. In this position, Mg 2+ additionally interacts with the carboxylate of a methyl donor cofactor S-adenosylmethionine (SAM). The computational results are validated by experimental mutation studies, which demonstrate the importance of the Mg 2+ -binding residues for the catalytic activity. The presence of Mg 2+ in the binding pocket induces SAM to adopt a unique bent shape required for the methyl transfer activity and causes a structural reorganization of the active site. Quantum mechanical calculations show that the methyl transfer is energetically feasible only when Mg 2+ is bound in the position revealed by the MD simulations, demonstrating that its function is to align the active site residues within the topological knot-fold in a geometry optimal for catalysis. The obtained insights provide the opportunity for developing a strategy of antibacterial drug discovery based on targeting of Mg 2+ -binding to TrmD.

Structural and functional characterization of TrmM in m6A modification of bacterial tRNA

“…The requirement of metal ions has not yet been reported for other RNA N 6 MTases. Meanwhile, the bacterial tRNA m 1 G37 MTase, TrmD, is dependent on the divalent metal ion, Mg 2+ 38,39 . The primary role of the ion is proposed to be optimizing the active site geometry and the conformation of SAM based on a molecular dynamics simulation and quantum mechanics study 39 .…”

“…Meanwhile, the bacterial tRNA m 1 G37 MTase, TrmD, is dependent on the divalent metal ion, Mg 2+ . 38,39 The primary role of the ion is proposed to be optimizing the active site geometry and the conformation of SAM based on a molecular dynamics simulation and quantum mechanics study. 39 For Mc_TrmM, metal binding may involve additional components, such as tRNA.…”

“…38,39 The primary role of the ion is proposed to be optimizing the active site geometry and the conformation of SAM based on a molecular dynamics simulation and quantum mechanics study. 39 For Mc_TrmM, metal binding may involve additional components, such as tRNA. Metals bound to tRNA sometimes contribute to the correct conformation or folding of polynucleotides.…”

Tied up in knots: Untangling substrate recognition by the SPOUT methyltransferases

“…Uniquely, the methyl transfer reaction by TrmD only occurs when a Mg 2+ is bound in the active site (84,85). A recent study demonstrated through combined molecular dynamics simulations, quantum mechanical studies, and mutagenesis/ enzyme activity assays that the essential Mg 2+ ion binds to a negatively charged pocket in the TrmD active site, causing structural changes that force SAM to adopt its bent conformation and align with active site residues in an optimal orientation for catalysis (84).…”

Developing and Assessing Nonbonded Dummy Models of Magnesium Ion with Different Hydration Free Energy References

“…Magnesium ion (Mg 2+ ) is of great importance in biology − and materials science. − It acts as a cofactor taking part in more than three hundred enzymatic reactions; Mg 2+ promotes, for instance, the phosphoryl transfer and renders assistance to ATP binding in protein kinases . It plays roles in nucleic acid systems as well via stabilizing specific RNA structures and assisting in recognition of binding partners and the mediation of catalytic processes. − Molecular simulation as a powerful tool has attracted considerable attention in exploring the molecular mechanism behind experimental studies on metal-containing systems. − Reliable prediction of the physicochemical properties of the Mg 2+ ion and Mg 2+ -involved complex structures requires accurate force field models and efforts to improve the model performance continue as well …”