Parametrization of 4′-DTMP

Trimethoprim derivatives were parametrized using the Force Field ToolKit (ffTK)³³ plugin implemented in Visual Molecular Dynamics (VMD).³⁴ The protocol used in our previous study²⁴ to parametrize TMP and TMP⁺ was followed herein for the TMP derivative. All quantum mechanical calculations necessary for this protocol (optimization of charges, bonds, angles, and dihedrals) were performed by Gaussian09.³⁵

Although it is known that TMP is neutral in water, it may also be in protonated form in the local protein environment depending on the mutant.³⁶ In our previous study, the simulations were conducted in the presence of both neutral and protonated TMP (TMP⁺), and the protonation state was determined according to the maximum stabilization of the drug in the active site of DHFR.²⁴ Similarly, both neutral and protonated states of 4′-DTMP structures were parametrized in this work, and the resulting parameters are given in Table S1. We find the protonation state of 4′-DTMP to be the same as TMP; i.e., it is protonated when bound both to the WT and to the mutant enzyme according to its hydrogen bonding profile (Figure S1). Therefore, in this study, only results from the protonated forms will be discussed.